Global longitudinal strain and plasma biomarkers for prognosis in heart failure complicated by diabetes: a prospective observational study.

BACKGROUND: Heart failure (HF) and diabetes are associated with increased incidence and worse prognosis of each other. The prognostic value of global longitudinal strain (GLS) measured by cardiovascular magnetic resonance (CMR) has not been established in HF patients with diabetes. METHODS: In this prospective, observational study, consecutive patients (n = 315) with HF underwent CMR at 3T, including GLS, late gadolinium enhancement (LGE), native T1, and extracellular volume fraction (ECV) mapping. Plasma biomarker concentrations were measured including: N-terminal pro B-type natriuretic peptide(NT-proBNP), high-sensitivity troponin T(hs-TnT), growth differentiation factor 15(GDF-15), soluble ST2(sST2), and galectin 3(Gal-3). The primary outcome was a composite of all-cause mortality or HF hospitalisation. RESULTS: Compared to those without diabetes (n = 156), the diabetes group (n = 159) had a higher LGE prevalence (76 vs. 60%, p < 0.05), higher T1 (1285±42 vs. 1269±42ms, p < 0.001), and higher ECV (30.5±3.5 vs. 28.8±4.1%, p < 0.001). The diabetes group had higher NT-pro-BNP, hs-TnT, GDF-15, sST2, and Gal-3. Diabetes conferred worse prognosis (hazard ratio (HR) 2.33 [95% confidence interval (CI) 1.43-3.79], p < 0.001). In multivariable Cox regression analysis including clinical markers and plasma biomarkers, sST2 alone remained independently associated with the primary outcome (HR per 1 ng/mL 1.04 [95% CI 1.02-1.07], p = 0.001). In multivariable Cox regression models in the diabetes group, both GLS and sST2 remained prognostic (GLS: HR 1.12 [95% CI 1.03-1.21], p = 0.01; sST2: HR per 1 ng/mL 1.03 [95% CI 1.00-1.06], p = 0.02). CONCLUSIONS: Compared to HF patients without diabetes, those with diabetes have worse plasma and CMR markers of fibrosis and a more adverse prognosis. GLS by CMR is a powerful and independent prognostic marker in HF patients with diabetes.

A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA.

Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.

Correction: Cserzo et al. The First Quarter Century of the Dense Alignment Surface Transmembrane Prediction Method. Int. J. Mol. Sci. 2023, 24, 14016.

There was an error in the original publication [...].

To kill or to be killed: pangenome analysis of Escherichia coli strains reveals a tailocin specific for pandemic ST131.

BACKGROUND: Escherichia coli (E. coli) has been one of the most studied model organisms in the history of life sciences. Initially thought just to be commensal bacteria, E. coli has shown wide phenotypic diversity including pathogenic isolates with great relevance to public health. Though pangenome analysis has been attempted several times, there is no systematic functional characterization of the E. coli subgroups according to the gene profile. RESULTS: Systematically scanning for optimal parametrization, we have built the E. coli pangenome from 1324 complete genomes. The pangenome size is estimated to be ~25,000 gene families (GFs). Whereas the core genome diminishes as more genomes are added, the softcore genome (≥95% of strains) is stable with ~3000 GFs regardless of the total number of genomes. Apparently, the softcore genome (with a 92% or 95% generation threshold) can define the genome of a bacterial species listing the critically relevant, evolutionarily most conserved or important classes of GFs. Unsupervised clustering of common E. coli sequence types using the presence/absence GF matrix reveals distinct characteristics of E. coli phylogroups B1, B2, and E. We highlight the bi-lineage nature of B1, the variation of the secretion and of the iron acquisition systems in ST11 (E), and the incorporation of a highly conserved prophage into the genome of ST131 (B2). The tail structure of the prophage is evolutionarily related to R2-pyocin (a tailocin) from Pseudomonas aeruginosa PAO1. We hypothesize that this molecular machinery is highly likely to play an important role in protecting its own colonies; thus, contributing towards the rapid rise of pandemic E. coli ST131. CONCLUSIONS: This study has explored the optimized pangenome development in E. coli. We provide complete GF lists and the pangenome matrix as supplementary data for further studies. We identified biological characteristics of different E. coli subtypes, specifically for phylogroups B1, B2, and E. We found an operon-like genome region coding for a tailocin specific for ST131 strains. The latter is a potential killer weapon providing pandemic E. coli ST131 with an advantage in inter-bacterial competition and, suggestively, explains their dominance as human pathogen among E. coli strains.

The First Quarter Century of the Dense Alignment Surface Transmembrane Prediction Method.

The dense alignment surface (DAS) transmembrane (TM) prediction method was first published more than 25 years ago. DAS was the one of the earliest tools to discriminate TM proteins from globular ones and to predict the sequence positions of TM helices in proteins with high accuracy from their amino acid sequence alone. The algorithmic improvements that followed in 2002 (DAS-TMfilter) made it one of the best performing tools among those relying on local sequence information for TM prediction. Since then, many more experimental data about membrane proteins (including thousands of 3D structures of membrane proteins) have accumulated but there has been no significant improvement concerning performance in the area of TM helix prediction tools. Here, we report a new implementation of the DAS-TMfilter prediction web server. We reevaluated the performance of the method using a five-times-larger, updated test dataset. We found that the method performs at essentially the same accuracy as the original even without any change to the parametrization of the program despite the much larger dataset. Thus, the approach captures the physico-chemistry of TM helices well, essentially solving this scientific problem.

Knockout of the non-essential gene SUGCT creates diet-linked, age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype.

SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as "crown-like" structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.

AllerCatPro-prediction of protein allergenicity potential from the protein sequence.

MOTIVATION: Due to the risk of inducing an immediate Type I (IgE-mediated) allergic response, proteins intended for use in consumer products must be investigated for their allergenic potential before introduction into the marketplace. The FAO/WHO guidelines for computational assessment of allergenic potential of proteins based on short peptide hits and linear sequence window identity thresholds misclassify many proteins as allergens. RESULTS: We developed AllerCatPro which predicts the allergenic potential of proteins based on similarity of their 3D protein structure as well as their amino acid sequence compared with a data set of known protein allergens comprising of 4180 unique allergenic protein sequences derived from the union of the major databases Food Allergy Research and Resource Program, Comprehensive Protein Allergen Resource, WHO/International Union of Immunological Societies, UniProtKB and Allergome. We extended the hexamer hit rule by removing peptides with high probability of random occurrence measured by sequence entropy as well as requiring 3 or more hexamer hits consistent with natural linear epitope patterns in known allergens. This is complemented with a Gluten-like repeat pattern detection. We also switched from a linear sequence window similarity to a B-cell epitope-like 3D surface similarity window which became possible through extensive 3D structure modeling covering the majority (74%) of allergens. In case no structure similarity is found, the decision workflow reverts to the old linear sequence window rule. The overall accuracy of AllerCatPro is 84% compared with other current methods which range from 51 to 73%. Both the FAO/WHO rules and AllerCatPro achieve highest sensitivity but AllerCatPro provides a 37-fold increase in specificity. AVAILABILITY AND IMPLEMENTATION: https://allercatpro.bii.a-star.edu.sg/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification and engineering of 32 membered antifungal macrolactone notonesomycins.

Notonesomycin A is a 32-membered bioactive glycosylated macrolactone known to be produced by Streptomyces aminophilus subsp. notonesogenes 647-AV1 and S. aminophilus DSM 40186. In a high throughput antifungal screening campaign, we identified an alternative notonesomycin A producing strain, Streptomyces sp. A793, and its biosynthetic gene cluster. From this strain, we further characterized a new more potent antifungal non-sulfated analogue, named notonesomycin B. Through CRISPR-Cas9 engineering of the biosynthetic gene cluster, we were able to increase the production yield of notonesomycin B by up to 18-fold as well as generate a strain that exclusively produces this analogue.

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

BACKGROUND: Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. RESULTS: Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. CONCLUSIONS: We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.

Prenylation of viral proteins by enzymes of the host: Virus-driven rationale for therapy with statins and FT/GGT1 inhibitors.

Intracellular bacteria were recently shown to employ eukaryotic prenylation system for modifying activity and ensuring proper intracellular localization of their own proteins. Following the same logic, the proteins of viruses may also serve as prenylation substrates. Using extensively validated high-confidence prenylation predictions by PrePS with a cut-off for experimentally confirmed farnesylation of hepatitis delta virus antigen, we compiled in silico evidence for several new prenylation candidates, including IRL9 (CMV) and few other proteins encoded by Herpesviridae, Nef (HIV-1), E1A (human adenovirus 1), NS5A (HCV), PB2 (influenza), HN (human parainfluenza virus 3), L83L (African swine fever), MC155R (molluscum contagiosum virus), other Poxviridae proteins, and some bacteriophages of human associated bacteria. If confirmed experimentally, these findings may aid in dissection of molecular functions of uncharacterized viral proteins and provide a novel rationale for statin and FT/GGT1-based inhibition of viral infections. Prenylation of bacteriophage proteins may aid in moderation of microbial infections.

Darkness in the Human Gene and Protein Function Space: Widely Modest or Absent Illumination by the Life Science Literature and the Trend for Fewer Protein Function Discoveries Since 2000.

The mentioning of gene names in the body of the scientific literature 1901-2017 and their fractional counting is used as a proxy to assess the level of biological function discovery. A literature score of one has been defined as full publication equivalent (FPE), the amount of literature necessary to achieve one publication solely dedicated to a gene. It has been found that less than 5000 human genes have each at least 100 FPEs in the available literature corpus. This group of elite genes (4817 protein-coding genes, 119 non-coding RNAs) attracts the overwhelming majority of the scientific literature about genes. Yet, thousands of proteins have never been mentioned at all, ≈2000 further proteins have not even one FPE of literature and, for ≈4600 additional proteins, the FPE count is below 10. The protein function discovery rate measured as numbers of proteins first mentioned or crossing a threshold of accumulated FPEs in a given year has grown until 2000 but is in decline thereafter. This drop is partially offset by function discoveries for non-coding RNAs. The full human genome sequencing does not boost the function discovery rate. Since 2000, the fastest growing group in the literature is that with at least 500 FPEs per gene.

Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin.

Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation.

Discovering novel SNPs that are correlated with patient outcome in a Singaporean cancer patient cohort treated with gemcitabine-based chemotherapy.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) can influence patient outcome such as drug response and toxicity after drug intervention. The purpose of this study is to develop a systematic pathway approach to accurately and efficiently predict novel non-synonymous SNPs (nsSNPs) that could be causative to gemcitabine-based chemotherapy treatment outcome in Singaporean non-small cell lung cancer (NSCLC) patients. METHODS: Using a pathway approach that incorporates comprehensive protein-protein interaction data to systematically extend the gemcitabine pharmacologic pathway, we identified 77 related nsSNPs, common in the Singaporean population. After that, we used five computational criteria to prioritize the SNPs based on their importance for protein function. We specifically selected and screened six candidate SNPs in a patient cohort with NSCLC treated with gemcitabine-based chemotherapy. RESULT: We performed survival analysis followed by hematologic toxicity analyses and found that three of six candidate SNPs are significantly correlated with the patient outcome (P < 0.05) i.e. ABCG2 Q141K (rs2231142), SLC29A3 S158F (rs780668) and POLR2A N764K (rs2228130). CONCLUSIONS: Our computational SNP candidate enrichment workflow approach was able to identify several high confidence biomarkers predictive for personalized drug treatment outcome while providing a rationale for a molecular mechanism of the SNP effect. TRIAL REGISTRATION: NCT00695994. Registered 10 June, 2008 'retrospectively registered'.

Charged residues next to transmembrane regions revisited: "Positive-inside rule" is complemented by the "negative inside depletion/outside enrichment rule".

BACKGROUND: Transmembrane helices (TMHs) frequently occur amongst protein architectures as means for proteins to attach to or embed into biological membranes. Physical constraints such as the membrane's hydrophobicity and electrostatic potential apply uniform requirements to TMHs and their flanking regions; consequently, they are mirrored in their sequence patterns (in addition to TMHs being a span of generally hydrophobic residues) on top of variations enforced by the specific protein's biological functions. RESULTS: With statistics derived from a large body of protein sequences, we demonstrate that, in addition to the positive charge preference at the cytoplasmic inside (positive-inside rule), negatively charged residues preferentially occur or are even enriched at the non-cytoplasmic flank or, at least, they are suppressed at the cytoplasmic flank (negative-not-inside/negative-outside (NNI/NO) rule). As negative residues are generally rare within or near TMHs, the statistical significance is sensitive with regard to details of TMH alignment and residue frequency normalisation and also to dataset size; therefore, this trend was obscured in previous work. We observe variations amongst taxa as well as for organelles along the secretory pathway. The effect is most pronounced for TMHs from single-pass transmembrane (bitopic) proteins compared to those with multiple TMHs (polytopic proteins) and especially for the class of simple TMHs that evolved for the sole role as membrane anchors. CONCLUSIONS: The charged-residue flank bias is only one of the TMH sequence features with a role in the anchorage mechanisms, others apparently being the leucine intra-helix propensity skew towards the cytoplasmic side, tryptophan flanking as well as the cysteine and tyrosine inside preference. These observations will stimulate new prediction methods for TMHs and protein topology from a sequence as well as new engineering designs for artificial membrane proteins.

Toward Allosterically Increased Catalytic Activity of Insulin-Degrading Enzyme against Amyloid Peptides.

The physiological role of insulin-degrading enzyme (IDE) in the intracytosolic clearance of amyloid ß (Aß) and other amyloid-like peptides supports a hypothesis that human IDE hyperactivation could be therapeutically beneficial for the treatment of late-onset Alzheimer's disease (AD). The major challenge standing in the way of this goal is increasing the specific catalytic activity of IDE against the Aß substrate. There were previous indications that the allosteric mode of IDE activity regulation could potentially provide a highly specific path toward degradation of amyloid-like peptides, while not dramatically affecting activity against other substrates. Recently developed theoretical concepts are used here to explore potential allosteric modulation of the IDE activity as a result of single-residue mutations. Five candidates are selected for experimental follow-up and allosteric free energy calculations: Ser137Ala, Lys396Ala, Asp426Ala, Phe807Ala, and Lys898Ala. Our experiments show that three mutations (Ser137Ala, Phe807Ala, and Lys898Ala) decrease the Km of the Aß substrate. Mutation Lys898Ala results in increased catalytic activity of IDE; on the other hand, Lys364Ala does not change the activity and Asp426Ala diminishes it. Quantifying effects of mutations in terms of allosteric free energy, we show that favorable mutations lead to stabilization of the catalytic sites and other function-relevant distal sites as well as increased dynamics of the IDE-N and IDE-C halves that allow efficient substrate entrance and cleavage. A possibility for intramolecular upregulation of IDE activity against amyloid peptides via allosteric mutations calls for further investigations in this direction. Ultimately, we are hopeful it will lead to the development of IDE-based drugs for the treatment of the late-onset form of AD characterized by an overall impairment of Aß clearance.

Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F.

The ability of the vancomycin-resistant Enterococcus faecalis (V583) to restore redox homeostasis via antioxidant defense mechanism is of importance, and knowledge into this defense is essential to understand its antibiotic-resistance and survival in hosts. The flavoprotein disulfide reductase AhpR, composed of the subunits AhpC and AhpF, represents one such vital part. Circular permutation was found to be a feature of the AhpF protein family. E. faecalis (V583) AhpF (EfAhpF) appears to be a representative of a minor subclass of this family, the typically N-terminal two-fold thioredoxin-like domain (NTD_N/C) is located at the C-terminus, whereas the pyridine nucleotide-disulfide oxidoreductase domain is encoded in the N-terminal part of its sequence. In EfAhpF, these two domains are connected via an unusually long linker region providing optimal communication between both domains. EfAhpF forms a dimer in solution similar to Escherichia coli AhpF. The crystallographic 2.3Å resolution structure of the NTD_N/C domain reveals a unique loop-helix stretch (409ILKDTEPAKELLYGIEKM426) not present in homologue domains of other prokaryotic AhpFs. Deletion of the unique 415PAKELLY421-helix or of 415PAKELL420 affects protein stability or attenuates peroxidase activity. Furthermore, mutation of Y421 is described to be essential for E. faecalis AhpF's optimal NADH-oxidative activity.

Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.

The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation. In previous studies, we were able to elucidate conformational flexibility of Escherichia coli AhpF during the catalytic cycle and its binding site, the N-terminal domain (NTD), to AhpC. We proposed the novel binding and release mechanism of EcAhpC-AhpF, which is mediated by the well defined redox-state linked conformational changes associated with the C-terminal tail and active site regions of EcAhpC. Here, we have proceeded further to elucidate the solution structure of E. coli AhpC and the stable ensemble formation with EcAhpF using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. The EcAhpC-AhpF complex structure with a stoichiometry of AhpC10:AhpF2 reveals that dimeric EcAhpF in its extended conformation enables the NTD disulphide centers to come in close proximity to the redox-active disulphide centers of EcAhpC, and provides an efficient electron transfer. Furthermore, the significance of the C-terminal tail of EcAhpC in ensemble formation is elucidated. SAXS data-based modeling revealed the flexible C-terminal tail of EcAhpC in solution, and its exposed nature, making it possible to contact the NTD of EcAhpF for stable complex formation.

Crystallographic and solution studies of NAD(+)- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps.

Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC. The NTD is connected to the C-terminal domain via a linker. Here, the first crystal structure of E. coli AhpF bound with NADH and NAD(+) has been determined at 2.5Å and 2.4Å resolution, respectively. The NADH-bound form of EcAhpF reveals that the NADH-binding domain is required to alter its conformation to bring a bound NADH to the re-face of the isoalloxazine ring of the flavin, and thereby render the NADH-domain dithiol center accessible to the NTD disulfide center for electron transfer. The NAD(+)-bound form of EcAhpF shows conformational differences for the nicotinamide end moieties and its interacting residue M467, which is proposed to represent an intermediate product-release conformation. In addition, the structural alterations in EcAhpF due to NADH- and NAD(+)-binding in solution are shown by small angle X-ray scattering studies. The EcAhpF is revealed to adopt many intermediate conformations in solution to facilitate the electron transfer from the substrate NADH to the C-terminal domain, and subsequently to the NTD of EcAhpF for the final step of AhpC reduction.

Single-residue posttranslational modification sites at the N-terminus, C-terminus or in-between: To be or not to be exposed for enzyme access.

Many protein posttranslational modifications (PTMs) are the result of an enzymatic reaction. The modifying enzyme has to recognize the substrate protein's sequence motif containing the residue(s) to be modified; thus, the enzyme's catalytic cleft engulfs these residue(s) and the respective sequence environment. This residue accessibility condition principally limits the range where enzymatic PTMs can occur in the protein sequence. Non-globular, flexible, intrinsically disordered segments or large loops/accessible long side chains should be preferred whereas residues buried in the core of structures should be void of what we call canonical, enzyme-generated PTMs. We investigate whether PTM sites annotated in UniProtKB (with MOD_RES/LIPID keys) are situated within sequence ranges that can be mapped to known 3D structures. We find that N- or C-termini harbor essentially exclusively canonical PTMs. We also find that the overwhelming majority of all other PTMs are also canonical though, later in the protein's life cycle, the PTM sites can become buried due to complex formation. Among the remaining cases, some can be explained (i) with autocatalysis, (ii) with modification before folding or after temporary unfolding, or (iii) as products of interaction with small, diffusible reactants. Others require further research how these PTMs are mechanistically generated in vivo.